Terpyridine derivatives

ABSTRACT

The invention pertains to terpyridine compounds having structure (I). These compounds form fluorescent lanthanide chelates with the appropriate metal ions. The fluorescent metal chelates are useful as probes in time-resolved fluorescence spectroscopy.

This is a continuation of application Ser. No. 466,414, filed Mar. 5,1990 now abandoned, and the benefits of 35 USC 120 are claimed relativeto it.

FIELD OF INVENTION

The invention pertains to organic complexing agents (ligands orchelators) having a 2,2':6',2" terpyridine structure. Throughout thisspecification compounds having this structure will be calledterpyridines, if not otherwise specified. The ligands of the inventionform fluorescent lanthanide chelates with the appropriate metal ions.

The new chelates as well as the ligand as such may find application inthose fields that are classical for chelates. The fluorescent metalchelates of our invention are useful as probes in time-resolvedfluorescence spectroscopy. Contrary to many other known lanthanidechelators, the chelators of this invention very often give stronglyfluorescent chelates with both Tb³⁺ and Eu³⁺. This might imply that theywill be very important in multi labeling techniques.

The sensitivity of the analytical methods based on molecularfluorescence is limited by the background signal due to either Ramanscattering or fluorescent impurities in the sample. Both of theseinterferences are generally short-lived phenomena, i.e. the radiativetransition following the excitation of the molecule occurs within amicrosecond time span. Thus any compound with a long-lived fluorescencecan be effectively determined in the presence of short-lived backgroundif a fluorometer with time resolution is at hand. In this fluorometerthe sample is illuminated by an intermittent light source such that thelong-lived fluorescence is measurable during the dark period subsequentto the decay of the short-lived background.

DESCRIPTION OF THE PRIOR ART

The chelating ability of terpyridines, bipyridines and substitutedpyridines are well known EP-A-68,875 (p. 29), Chemical Abstracts 98(1983) 226913k, 102 (1985) 71589j, 103 (1985) 71419z and 106 (1987)94885, FR-A-2,570,703, Chem Berichte 118 (1985) 212-7, EP-A-195,413 andEP-A-203,047).

Time resolved fluorescence spectroscopy by the use of fluorescentlanthanide chelates has also been known for some time. The methodspresented in DE-A-2,628,158 and U.S. Pat. No. 4,374,120 make use ofbeta-diketones as ligands in fluorescent lanthanide chelates used influoroimmunoassays. In DE-A-2,628,158 the light absorbing moiety of thefluorescent chelate is covalently conjugated to an antigen or antibodywhich during the test procedure is allowed to form an immune complexwith its immunological counterpart. In this type of assays the testprotocol is arranged so that the amount of the immune complex formedwill constitute indication means for the substance that is to bedetected (=analyte). The lanthanide chelate-labeled immune reactant usedhas an epitope in common with the analyte (analyte analogue) or is anantibody active component directed against the analyte or against otherantigenic or haptenic material used for the formation of the immunecomplex. In many versions of this type of assays the chelate may beconjugated directly to a molecule employed as an antigen or hapten.There are several different ways of categorizing fluoroimmunoassays andother assays employing biospecific affinity reactions (reactants). If,before the fluorescence measurement, the chelate incorporated in theimmune complex is separated from the portion of the chelate that is notincorporated, the methods are called heterogenous. If no such separationstep is included they are called homogenous. These two variants putdifferent demands on the lanthanide chelate used. The homogenous onesrequire that the chelate changes its fluorescent properties in ameasurable manner as a consequence of the immune complex formation,while no such requirement is needed for the heterogenous versions.Immunoassays are just one example of methods that utilize biospecificaffinity reactions. Analogously there are assay systems utilizing othertypes of biospecific affinity such as between DNA--complementary DNA,Protein A--Fc-part of IgG, lectin--carbohydrate structure etc. Theprinciples and classification outlined above apply equally well to them.

The major short-coming of the fluorescent lanthanide chelates is thatthey rarely combine good aqueous stability with efficient fluorescence.In aqueous solvents their fluorescence is often quenched by water.

In EP-A-64,484 a version of a lanthanide chelate heterogenousfluoroimmunoassay is given that has no requirements for labels that aregood fluorescers. Instead the interest is focused on the aqueousstability of the lanthanide chelate used. The ligand only serves tocarry the lanthanide through the separation step, after which thelanthanide cat-ion is dissociated at a low pH and a highly fluorescentchelate is formed with an aromatic beta-diketone as ligand in a micellarphase. This method gives a very good sensitivity but suffers from asomewhat lengthy procedure.

It would be very advantageous to have highly fluorescent lanthanideprobes with good aqueous stability. Such probes would allow homogenousassays and shorter assay procedures in heterogenous assays. They wouldalso find use in other fluorescence methods, e.g. fluorescencemicroscopy.

Lanthanide chelates fluorescing in aqueous media have previously beensuggested (EP-A-180,492 (macropolycyclic), EP-A-68,875 (phenols and arylethers and one specific terpyridine chelator), EP-A-195,413 (arylpyridines) and EP-A-203,047 (ethynyl pyridines)). Although they to somedegree can compensate for the short-comings set forth above onlylanthanide chelates of the ethynyl pyridines (EP-A-203,047)have turnedout to exhibit an efficient quantum yield and an aqueous stability forthe commercialisation as directly fluorescing labels.

THE INVENTION

The compounds of the invention have the common structure given informula I and comprise also ordinary analogues thereof, such as acid,ester, salt and chelate forms involving one or more of their chelatingheteroatoms. ##STR1##

In formula I, n is an integer 0, 1, or 2, preferably 1, and specifiesthat the group X-Y is a substituent replacing a hydrogen or a group X₁-X₅ in the parent terpyridine compound.

X₁ -X₅ are selected from

a) hydrogen

b) hydrocarbon group which may be straight, branched or cyclic, such asalkyl, alkenyl, alkynyl and an aromatic group (aryl), and containingadditional structures not participating in the chelation such asaromatic ring systems, ether, thioether, ester, amido, amino (primary,secondary or tertiary), carboxy, halo, cyano etc., and other structuresexhibiting heteroatoms,

c) cyano, halo and nitro, and

d) carboxylic acid (COOH), amido (CONH₂), amino (NH₂), hydroxy (OH),mercapto (SH) and substituted forms of these groups in which hydrogenhas been replaced with a hydrocarbon group as defined in b) above, andthe hydrogen of the amino and hydroxy group optionally also having thepossibility of being replaced with an acyl group, RCO, in which R is anhydrocarbon group as defined in b) above.

Examples of alkyl are lower alkyl having less than 12, such as 1-5,carbon atoms.

Examples of different aromatic groups are phenyl, quinolyl, naphthyl,pyridyl, pyrimidyl etc. all of which may contain additionalsubstituents. By selecting alkenyl, alkynyl or aromatic system directlybound or bound via a carbonyl, or via an uncharged nitrogen atom toanyone of the heteroaromatic rings in formula I, the conjugated electronsystem can be extended to more aromatic rings or carbon atoms.Preferably at most two aromatic ring structures are conjugated to theterpyridine structure.

The preferred X₁ -X₅ are hydrogen, hydroxyl, alkoxyl, amino, amido,alkyl, such as aminoalkyl, alkylthio, alkylamino, alkynyl, such asaminoethynyl, aryl, such as aminoaryl, aralkyl, aryloxyl, arylamino andarylethynyl, Fenyl(ene) is the preferred aryl moiety. From the syntheticpoint of view both X₁ and X₅ are preferably hydrogen.

Z₁ and Z₂ are chelating groups, that may be identical or different.Preferably Z₁ and Z₂ are --CH₂ --Z' and --CH₂ --Z", respectively, havingthe chelating ability confined to Z' and Z".

Each of Z₁ and Z₂ comprises two or more heteroatoms having a free pairof electrons and placed at a distance of two or three atoms from eachother. Examples of efficient chelating heteroatoms are amino nitrogenatoms (primary, secondary and tertiary amines), negatively chargedoxygen atoms e.g. in carboxylate anions (COO⁻), enolate anions(C═C--O⁻)phosphates or phosphonates. Another good chelating structure isthe hydroxamate group (CONOH). In most cases the bridge between twoneighbouring chelating heteroatoms contains 1, 2 or 3 aliphatic carbonatoms. Among particularly important Z' and Z" structures may bementioned N-biscarboxymethyl amino and N-biscarboxyethyl amino groups,the analogous phosphate (--N(--CH₂ --O--PO₃ ²⁻)₂) and phosphonate(--N(--CH₂ --PO₃ ²⁻)₂) and 2,6dicarboxypiperidin-1-yl. Alternatively, Z₁and Z₂ may form a bridge connecting the two outer pyridine rings givinga macrocyclic chelating compound. Preferably such a bridge is --CH₂N(CH₂ COO⁻)CH₂ --(Z'=Z"=NCH.sub. 2 COO⁻).

In some compounds of the invention the chelating heteroatoms (N and O)may exist as the corresponding protonated forms and for O also as esterforms, such as lower alkyl (C₁ -C₆) or benzyl esters.

From the spectrofluorometric point of view Z and Z' are preferablychelating to Eu³⁺, Tb³⁺, Dy³⁺ or Sm³⁺.

X-Y represents an inert organic group in which X is an inert and stablebridge and Y is (a) a functional group or (b) a residue of an organiccompound (Y') that has properties retained in the compound of formula I(n=1 or 2) after it has been coupled covalently to the parent compound.The term "inert" above means that the group or bridge characterized bythis adjective does not have any efficient chelating heteroatom closerthan at a distance of four atoms from the heteroatoms participating inthe chelation of a joint metal ion. In actual practice this means thatthe four atoms are normally represented by a four-carbon chain. By"stable" is meant that the bridge X does not deteriorate when thecompounds of the invention are used, for instance the bridge does noteasily undergo hydrolysis. In formula I, X-Y exist as a substituentreplacing a hydrogen in a Z₁ and/or Z₂ group, preferably in a Z' and/orZ" group, or a hydrogen in one or two X₁ -X₅.

X may contain at least one structural element selected from among thefollowing: --NR-- (secondary and tertiary amine), --CONR-- and --NRCO--(substituted amide), --S--S-- (aliphatic disulfide), --S-- (aliphaticthioether), --O-- (ether), --COO-- and --OOC-- (ester), --N═N-- (diaza)and pure hydrocarbon chain which may be straight, branched or cyclic andcontain from 1 to 12 carbon atoms. The carbon chain may be purelyaliphatic or purely aromatic (including phenyl, naphthyl, quinolyl,pyridyl and bipyridyl), and other inert functional groups notparticipating in the chelation mentioned above. The symbol R in thesubstituted amide above represents preferably hydrogen but may be alkyl,for instance an alkyl having less than 5 carbon atoms.

Y may be selected from two main categories (A and B below):

A) Y may be the residue of an organic compound (Y') having certainproperties which are substantially retained in the compound of formula I(n=1 or 2). The compound (Y') may be a biologically active moleculehaving the ability to participate in biospecific affinity reactions,such as between antigens (haptens) and the homologous antibody activecomponents, nucleotides, aligonucleotides, complementary nucleic acids(RNA, DNA), lectins and carbohydrate structures, protein A and IgG etc.This type of biologically active molecules are often called targetingsubstances (targeting molecules). Usually they have been or can beeasily derivatized to contain functional groups permitting conjugationto diversified types of compounds. The compound (Y') may also be amultifunctional organic compound being bound by one of its functionalgroups to the bridge X so as to leave at least one of its remainingfunctional groups free for further derivatization.

B) Y may be a functional group so selected that it can be made to reactchemically with a functional group A of an organic compound (Y') so asto form a covalent linkage between Y' and a compound of formula I (n=1or 2). The selection of Y deepens on A and vice versa, but it isbelieved that any artisan can make the proper selection of mutuallyreactive groups. Y and A may be selected from among electrophilic andnucleophilic groups. If they are a pair of electrophilic groups or apair of nucleophilic groups, it is possible for instance to (a) employoxidative coupling for forming the bond (e.g. --SH+HS----S--S--) or (b)convert one of the groups of the pair chemically to a group of theopposite type. An example of the latter case is the activation withbifunctional coupling reagents (also called activation reagents). If Yis nucleophilic and A electrophilic or vice versa these two groups canusually be reacted with each other without any preceding activation.Most nucleophilic groups comprise a heteroatom having an electron pairavailable for reaction with an electron deficient atom (electrophilicgroup).

Examples of suitable functional groups include isothiocyanato,bromoacetamido, iodoacetamido, succinamido, pyridyldithio, mercapto,cargoxyl and its active esters (e.g. N-hydroxysuccinimido orp-nitrophenyl), hydroxyl, aldehyde, amino, diazonium, tosyl, mesytylyl,trexyl, phosphodiester or phosphotriester. Other functional groups areknown to those skilled in the art.

In a compound according to the invention it is imperative that all thegroups mentioned can coexist. However, the reactive group Y does notnecessarily have to coexist with a chelating group of the invention. Forsome purposes the chelating part of the molecule may be temporarilyprotected e.g. in the form of an ester so that the protected ligand willbe coupled to the target molecule, and after deblocking may finally formthe desired labelled product. The protective group is chosen inaccordance with known principles (see for instance Protective Groups inOrganic Synthesis; Greene, Tenn.; John Wiley & Sons Inc; USA (1981).Factors to be taken into account when the group is chosen are inter aliathe stability of the compound with which Y is to be reacted, thereactivity of Y and the type of structure formed upon reaction of Y andthe compound intended.

The examples of this specification given wellknown methods forintroducing functionalized methylene groups next to a pyridine nucleus.These groups can subsequently be reacted with iminobisacetic esters tothe formation of chelating groups Z₁ and Z₂. Phosphonate groups can beintroduced according to J. Org. Chem. 31 (1966) 1603-- and phosphategroups according to Helv. Chim. Acta 70 (1987) 175. For the formation ofchelates with different metal ions, see Nucl. Med. Biol. 13 (1986) 311--and references cited therein. Prior to the first filing of this patentapplication no publication had presented any good method for thesynthesis of iminobisacetic esters derivatives having a functionalizedsubstituent at the methylene group of their acetyl moiety. A syntheticroute has been developed at Wallac Oy, Finland and it will beexemplified in this specification in detail (examples 37-45. The sosynthesized derivatives may be reacted with 6,6"-bishalomethylterpyridines analogously to the method given in examples 34-35 below.

The preferred compounds of the invention have been summarized in formulaII. ##STR2##

In formula II, X₁ -X₅, n, X and Y have the same meaning as statedbefore, except that at most two of X₁ -X₅ are groups other thanhydrogen.

--------- means that X-Y is a substituent replacing a group X₁ -X₅ orH'. Ch is a chelating group selected from --COO⁻, --OPO₃ ²⁻, and --PO₃²⁻. This preferred aspect of the invention also comprises the acid,ester, salt and chelate forms as defined above.

In general terms one mode of our invention is to apply our newfluorescing chelates as probes in time-resolved fluorescentspectrometry. In one aspect the chelating light-absorbing moiety of thechelate is used as a fluorescence enhancer for methods in whichsubstantially non-fluorescent Eu³⁺ --, Dy³⁺ --, Sm³⁺ -- or Tb³⁺ --chelates are used. See for instance EP-A-64,484 (Wallac Oy) and U.S.Pat. No. 4,352,751 (Wieder). Another aspect is a time-resolvedfluorometric method using a fluorescing lanthanide chelate as the probefor the determination of a target substance (structure). Generally theseassay methods comprise two steps (i) marking specifically the targetstructure with a lanthanide (probe) chelate of this invention (or justthe chelating ligand if the target is a lanthanide ion forming afluorescing chelate with the ligant), and (ii) measuring by methodsknown per se the fluorescence related to said probe. Assay systemsemploying biospecific affinity reactions as given in the introductorypart are one of the most important systems falling under thisdefinition. Accordingly other steps may also be included and beperformed before, between or after steps (i) and (ii). Compare also U.S.Pat. No. 4,058,732 (Wieder).

The invention is defined in the appended claims which are part of thedescription. The invention will now be illustrated with the synthesis ofseveral compounds of the invention.

The structures and the synthetic routes employed in the experimentalpart are shown in reaction schemes 1-3. Schemes 1 and 2 illustrateterpyridine compounds having a 4'-respective 4- and 4"-substitutent(s).By the proper selection of R¹ in the starting compounds substituentswill be introduced permitting either direct coupling or coupling by theuse of common bifunctional coupling reagents to a biological activemolecule participating in biospecific affinity reactions. Examples ofsuch groups are nucleophilic groups (primary amino, isothiocyanato etc).Scheme 3 illustrates the synthesis of iminobisacetic acid derivativesthat can be used for the formation of the chelating groups Z₁ and Z₂ (offormula I). The derivatives contain a reactive group that cannotparticipate in the chelating but that can be used to link covalently abiological active molecule of the type as set forth above to theterpyridine structure. ##STR3##

EXAMPLE 1 1-(2-Pyridyl)-3-(3-nitrophenyl)-2-propenone, 3b

2-Acetylpyridine (1.82 g, 15.0 mmol) and 3-nitrobenzaldehyde (2.27 g,15.0 mmol) were added to a solution of KOH (0.67 g, 12.0 mmol) in water(6 ml) and methanol (54 ml). The solution was stirred at roomtemperature for 2 hours, whereafter the precipitation of the product wascomplete. The precipitate was filtered and washed with methanol.

Yield: 93%

¹ H NMR (CDCl₃): 7.52-7.55 (1H, m); 7.62 (1H, t, J=8 Hz); 7.91 (1H, dt,J=8 Hz and 2 Hz); 7.94 (1H, d, J=16 Hz); 8.01 (1H, d, j=8 Hz); 8.21 (1H,d, J=8 Hz); 8.26 (1H, dd, J=8 Hz and 2 Hz); 8.44 (1H, d, J=16 Hz); 8.59(1H, s); 8.78 (1H, d, J=5 Hz).

EXAMPLE 2 1-(2-Pyridyl)-3-(4-nitrophenyl)-2-propenone, 3a

This compound was synthesized using a method analogous to the synthesisdescribed in example 1, 3b. The crude product was crystallized fromethanol.

Yield: 68%

¹ H NMR (CDCl₃): 7.52-7.56 (1H, m); 7.87 (2H, d, J=9 Hz); 7.91 (1H, dt,J=8 Hz and 2 Hz), 7.92 (1H, d, J=16 Hz); 8.21 (1H, d, J=8 Hz); 8.27 (2H,d, J=9 Hz); 8.43 (1H, d, J=16 Hz); 8.76 (1H, d, J=5 Hz)

EXAMPLE 3 1-(2-Pyridylcarbonylmethyl)pyridinium iodide, 5

2-Acetylpyridine (1.21 g, 10.0 mmol) and iodine (2.54 g, 10.0 mmol) wereheated to reflux in pyridine (12 ml) for 1 hour. The solution wascooled, and the dark precipitate was filtered and washed with pyridine.

Yield: 72%

¹ H NMR (DMSO): 6.51 (2H, s); 7.82-7.85 (1H, m); 8.08 (1H, d, J=7 Hz);8.14 (1H, dt, J=8 and 2 Hz); 8.28 (2H, t, J=8 Hz); 8.73 (1H, t, J=8 Hz);8.87 (1H, d, J=5 Hz); 9.00 (2H, d, J=6 Hz).

EXAMPLE 4 4'-(3-Nitrophenyl)-2,2';6',2"-terpyridine, 6c

1-(2-Pyridyl)-3-(3-nitrophenyl)-2-propenone (2.54 g, 10.0 mmol) and1-(2-pyridylcarbonylmethyl)pyridinium iodide (3.26 g, 10.0 mmol) andammonium acetate (4.82 g, 60.0 mmol) were heated to reflux in aceticacid (37 mol) for 1.5 hours whereafter the solution was cooled and theprecipitate was filtered and washed with acetic acid. The crude productwas crystallized from acetonitrile.

Yield: 56%

¹ H NMR (DMSO): 7.54-7.58 (2H, m); 7.89 (1H, t, J=8 Hz); 8.06 (2H, dt,J=8 Hz and 2 Hz); 8.38-8.41 (1H, m); 8.41-8.44 (1H, m); 8.66 (1H, t, J=2Hz); 8.70 (2H, d, J=8 Hz); 8.78-7.80 (4H, m).

EXAMPLE 5 4'-(4-Nitrophenyl)-2,2';6',2"-terpyridine, 6b

This compound was synthesized using a method analogous to the synthesisdescribed in example 4, 6c. The crude product was purified with flashchromatography (silica, chloroform with methanol gradient).

Yield: 56%

¹ H NMR (DMSO): 7.53-7.56 (2H, m); 8.05 (2H, dt, J=8 Hz and 2 Hz); 8.22(2H, d, J=9 Hz); 8.40 (2H, d, J=9 Hz); 8.68 (2H, d, J=8 Hz); 8.76 (2H,s); 8.77 (2H, d, J=5 Hz).

EXAMPLE 6 2,2';6',2"-Terpyridine-N,N"-dioxide, 7a

2,2':6',2"-Terpyridine (0.94 g, 4.0 mmol) was dissolved indichloromethane (80 ml) and m-chloroperbenzoic acid (2.60 g, 15.1 mmol)was added. After stirring overnight some dichloromethane was added andthe solution was washed with 10% sodium carbonate. The dichloromethanephase was evaporated and purified with flash chromatography (silica,chloroform with methanol gradient).

Yield: 93%

¹ H NMR (CDCl₃): 7.31 (2H, dd, J=2 and 7 Hz); 7.40 (2H, dd, J=1 and 8Hz); 7.98 (1H, t, J=8 Hz); 8.20 (2H, dd, J=2 and 8 Hz); 8.37 (2H, dd,J=1 and 7 Hz); 8.94 (2H, d, J=8 Hz).

UV (λ_(max) in ethanol): 241, 279.

EXAMPLE 7 4'-(4-Nitrophenyl)-2,2';6',2"-terpyridine-N,N"-dioxide, 7b

This compound was synthesized using a method analogous to the synthesisdescribed in Example 6, 7a. The crude product was purified bycrystallization from a mixture of acetonitrile and methanol.

Yield: 51%

¹ H NMR (DMSO): 7.52-7.58 (4H, m); 8.11 (2H, dd, J=7 Hz and 2 Hz);8.26-8.28 (2 H, m); 8.41-8.46 (4H, m); 9.16 (2H, s).

EXAMPLE 8 4'-(3-Nitrophenyl)-2,2';6',2"-terpyridine-N,N"-dioxide, 7c

This compound was synthesized using a method analogous to the synthesisdescribed in Example 6, 7a. The crude product was purified bycrystallization from a mixture of ethanol and chloroform.

Yield: 78%

¹ H NMR (DMSO): 7.52-7.58 (4H, m); 7.89 (1H, t, J=8 Hz); 8.25-8.28 (2H,m); 8.31 (1H, d, J=8 Hz); 8.38 (1H, dd, J=8 Hz and 1 Hz); 8.43-8.45 (2H,m); 8.61 (1H, t, J=2 Hz); 9.17 (2H, s).

EXAMPLE 9 6,6"-Dicyano-2,2';6',2"-terpyridine, 8a

2,2';6',2"-Terpyridine-N,N"-dioxide (7a) (0.96 g, 3.6 mmol) wassuspended in dichloromethane (10 ml). N,N-dimethylcarbamyl chloride(0.95 g, 8.8 mmol) and trimethylsilyl cyanide (1.26 g, 12.7 mmol) wereadded. After stirring for two weeks at room temperature, 10% potassiumcarbonate was added until the solution was neutral. The phases wereseparated and the water phase was extracted with chloroform. Thechloroform was evaporated and the residue was crystallized from amixture of acetonitrile and tetrahydrofurane.

Yield: 65%

¹ H NMR (CDCl₃): 7.75 (2H, d, J=8 Hz); 8.01 (2H, t, J=8 Hz); 8.05 (1H,t, J=8 Hz); 8.58 (2H, d, J=8 Hz); 8.82 (2H, d, J=8 Hz)

UV (λ_(max) in ethanol): 215, 250, 291.

EXAMPLE 10 4'-(4-Nitrophenyl)-6,6"-dicyano-2,2';6',2"-terpyridine, 8b

This compound was synthesized using a method analogous to the synthesisdescribed in Example 9, 8a. The crude product was purified bycrystallization from a mixture of acetonitrile and tetrahydrofurane.

Yield: 56%

¹ H NMR (DMSO): 8.22 (2H, dd, J=8 Hz and 1 Hz); 8.30 (2H, d, J=9 Hz);8.33 (2H, t, J=8 Hz); 8.43 (2H, d, J=9 Hz); 8.77 (2H, s); 8.01 (2H, dd,J=8 Hz and 1 Hz).

EXAMPLE 11 4'-(3-Nitrophenyl)-6.6"-dicyano-2,2';6',2"-terpyridine, 8c

This compound was synthesized using a method analogous to the synthesisdescribed in Example 9, 8a. The crude product was purified bycrystallization from a mixture of acetonitrile and tetrahydrofurane.

Yield: 71%

¹ H NMR (DMSO): 7.90 (1H, t, J=8 Hz); 8.22 (2H, dd, J=8 Hz and 1 Hz);8.33 (2H, t, J=8 Hz); 8.41-8.45 (1H, m); 8.47-8.72 (1H, m); 8.72 (1H,s); 8.77 (2H, s); 9.00 (2H, d, J=8 Hz).

EXAMPLE 12 6,6"-Bis(aminomethyl)-2,2';6',2"-terpyridinepentahydrochloride, 9a

Compound 8a (0.55 g, 1.9 mmol) was suspended in dry tetrahydrofurane (15ml). The suspension was bubbled with nitrogen. Borane tetrahydrofuranecomplex (1M, 25 ml, 25.0 mmol) was slowly added. After stirringovernight, the extra borane was destroyed by adding methanol. Themixture was evaporated to dryness and the residue was dissolved inethanol (30 ml) saturated with hydrochloric acid. After stirring for onehour the solution was cooled and the product was filtrated.

Yield: 54%

¹ H NMR (D₂ O): 4.68 (4H, s); 7.89 (2H, d, J=8 Hz); 8.31 (2H, t, J=8Hz); 8.51 (2H, d, J=8 Hz); 8.82 (2H, d, J=8 Hz); 8.90 (1H, t, J=8 Hz)

UV (λ_(max) in H₂ O): 232, 293

EXAMPLE 134'-(4-Nitrophenyl)-6,6"-bis(aminomethyl)-2,2';6',2"-terpyridinepentahydrochloride, 9b

This compound was synthesized using a method analogous to the synthesisdescribed in Example 12, 9a. The product was not purified and thereforeno proper NMR data was taken. The crude product was used as such in thenext step.

Yield: 56% of crude product

EXAMPLE 144'-(3-Nitrophenyl)-6,6"-bis(aminomethyl)-2,2';6',2"-terpyridinepentahydrochloride, 9c

This compound was synthesized using a method analogous to the synthesisdescribed in Example 12, 9a. The crude product was used in the next stepwithout further purifications.

Yield: 61% of crude product

EXAMPLE 156,6"-Bis[N,N-bis(t-butoxycarbonylmethyl)aminomethyl]-2,2';6',2"-terpyridine,10a

Compound 9a (0.43 g, 0.9 mmol), acetonitrile (15 ml), dry sodiumcarbonate (2.00 g) and t-butyl ester of bromoacetic acid (1.44 g, 7.4mmol) was refluxed for two hours and the salts were filtered. Theorganic phase was evaporated and purified with flash chromatography(silica, chloroform).

Yield: 38%

¹ H NMR (CDCl₃): 1.48 (36H, s); 3.56 (8H, s); 4.15 (4H, s); 7.66 (2H, d,J=8 Hz); 7.84 (2H, t, J=8 Hz); 7.90 (1H, t, J=8 Hz); 8.47 (2H, d, J=8Hz); 8.50 (2H, d, J=8 Hz)

UV (λ_(max) in ethanol): 235, 288.

EXAMPLE 164'-(4-Aminophenyl)-6,6"-bis[N,N-bis(ethoxycarbonylmethyl)aminomethyl]-2,2';6',2"-terpyridine,10b

The corresponding nitrocompound was synthesized from 9b and an ethylester of bromoacetic acid using a method analogous to the synthesisdescribed in 10a. The product was purified with flash chromatography(silica, chloroform with methanol gradient).

Yield: 20%

¹ H NMR (CDCl₃): 1.25 (12H, t, J=7 Hz); 4.18 (8H, q, J=7 Hz); 3.71 (8H,s); 4.23 (4H, s); 7.66 (2H, dd, J=8 Hz and 2 Hz); 7.90 (2H, t, J=8 Hz);8.06 (2H, d, J=9 Hz); 8.42 (2H, d, J=9 Hz); 8.57 (2H, dd, J=8 Hz and 2Hz); 8.76 (2H, s).

The aminocompound 10b was synthesized from the correspondingnitrocompound using a method analogous to the synthesis described in10c.

Yield: 16%

EXAMPLE 174'-(3-Aminophenyl)-6,6"-bis[N,N-bis(ethoxycarbonylmethyl)aminomethyl]-2,2';6',2"-terpyridine, 10c

The corresponding nitrocompound was synthesized from 9c and an ethylester of bromoacetic acid using a method analogous to the synthesisdescribed in 10a. The product was purified with flash chromatography(silica, chloroform with methanol gradient).

Yield: 53%

¹ H NMR (CDCl₃): 1.24 (12H, t, J=8 Hz); 4.17 (8H, q, J=8 Hz); 3.69 (8H,s); 4.23 (4H, s); 7.67 (2H, dd, J=8 Hz and 2 Hz); 7.89 (2H, t, J=8 Hz);7.85-8.30 (4 H, m); 8.57 (2H, dd, J=8 Hz and 2 Hz); 8.75 (2H, s).

The aminocompound 10c was synthesized from the correspondingnitrocompound (50 mg, 0.7 mmol) which was dissolved in methanol (2 ml).10% Palladium on carbon (10 mg) was added followed by slow addition ofsodium borohydride (4 mg, 0.1 mmol). After one hour the reaction mixturewas filtered and the solution was evaporated. The residue was dissolvedin 0.1M sodium hydroxide solution and the product was extracted withchloroform from the water phase. The organic phase was dried andevaporated. The product was purified with flash chromatography (CHCl₃,MeOH; 10:0.5).

Yield: 79%

EXAMPLE 186,6"-Bis[N,N-bis(carboxymethyl)aminomethyl]-2,2';6',2"-terpyridine, 11a

Tetraester (10a) (260 mg, 0.4 mmol) was dissolved in trifluoroaceticacid (3 ml). After two hours the trifluoroacetic acid was evaporated,some diethyl ether was added and the product was filtered.

Yield: 87%

¹ H NMR (DMSO): 3.99 (8H, s); 4.47 (4H, s); 7.65 (2H, d, J=8 Hz); 8.10(2H, t, J=8 Hz); 8.11 (1H, t, J=8 Hz); 8.48 (2H, d, J=8 Hz); 8.61 (2H,d, J=8 Hz)

UV (λ_(max) in water): 237, 288, 305.

EXAMPLE 194'-(4-Aminophenyl)-6,6"-bis[N,N-bis(carboxymethyl)aminomethyl]-2,2';6',2"-terpyridine,11b

This compound was synthesized using a method analogous to the synthesisdescribed in Example 20, 11c.

Yield: 100%

¹ H NMR (DMSO): 3.82 (8H, s); 4.31 (4H, s); 6.90 (2H, d, J=7 Hz); 7.67(2H, d, J=8 Hz); 7.80 (2H, d, J=9 Hz); 8.08 (2H, t, J=8 Hz); 8.60 (2H,d, J=8 Hz); 8.67 (2H, s).

EXAMPLE 204'-(3-Aminophenyl)-6,6"-bis[N,N-bis(carboxymethyl)-aminomethyl]-2,2';6',2"-terpyridine,11c

The corresponding tetraester, 10c (0.10 g, 0.17 mmol) was dissolved in 5ml 0.5M KOH-EtOH solution. After 2 hours stirring at room temperature,the ethanol was evaporated to dryness and water was added. The mixturewas stirred at room temperature for 30 min whereafter the product wasprecipitated with 2M hydrochloric acid.

Yield: 100%

¹ H NMR (DMSO): 3.74 (8H, s); 4.27 (4H, s); 7.71 (2H, d, J=7 Hz); 7.87(1H, t, J=8 Hz); 8.09 (2H, t, J=7 Hz); 8.33 (1H, d, J=8 Hz); 8.62 (1H,d, J=8 Hz); 8.63 (2H, d, J=7 Hz); 8.79 (2H, s); 8.83 (1H, s).

EXAMPLE 21 1-(2,6-Pyridyldicarbonylmethyl)-bispyridinium iodide, 13

2,6-Diacetylpyridine (8.16 g, 50.0 mmol) and iodide (25.38 g, 100.0mmol) were heated to reflux in pyridine (125 ml) for 2 hours. Thesolution was cooled, and the brown crystalline pyridinium salt (13)separated by filtration.

Yield: 87%

EXAMPLE 22 6,6"-Dicarboxy-4,4"-diphenyl-2,2';6',2"-terpyridine, 16a

A solution of 13 (2.86 g, 5.0 mmol), 2-oxo-4-phenyl-3-butenoic acid(1.76 g, 10.0 mmol) and ammonium acetate (8.86 g, 115.0 mmol) in aceticacid (50 ml) was heated to reflux for 5 hours, whereupon the brownsolution as cooled. The solid material was filtered and dried in air.The ammonium salt of 2 was dissolved in water and acidified with 6M HCl(pH about 1). The product was filtered and washed with water.

Yield: 34%

m.p. 279° C. (dec.).

¹ H NMR (DMSO): 7.56-8.06 (10H, m); 8.27 (1H, t); 8.40 (2H, d); 8.70(2H, d); 9.18 (2H, d).

IR (KBr): 1760, 1715, 1695, 1420, 1365, 1272 cm⁻¹ (C═O and C--O)

EXAMPLE 236,6"-Dicarboxy-4,4"-bis(4-nitrophenyl)-2,2';6',2"-terpyridine, 16b

Compound 16b was synthesized from 13 and 15b using a method analogous tothe synthesis described in 16a.

Yield: 57%

¹ H NMR (DMSO): 8.24 (1H, t); 8.28 (4H, d); 8.37 (4H, d); 8.43 (2H, d);8.65 (2H, d); 9.13 (2H, d).

EXAMPLE 24 6,6"-Dicarboxy-4,4"-bis(3-nitrophenyl)-2,2';6'2"-terpyridine,16c

Compound 16c was synthesized from 13 and 15c using a method analogous tothe synthesis described in 16a.

Yield: 81%

EXAMPLE 256,6"-Bis(methoxycarbonyl)-4,4"-diphenyl-2,2';6'2"-terpyridine, 17a

One drop of concentrated sulfuric acid was added to a suspension of 16a(0.77 g, 1.6 mmol) in methanol (5 ml). The mixture was heated to refluxfor 24 hours. The cold mixture was filtered ad the product wascrystallized from dichloromethane.

Yield: 61%

m.p. 285.5°-286.5° C.

¹ H NMR (CDCl₃): 4.13 (6H, s); 7.49-7.88 (10H, m); 8.07 (1H, t); 8.44(2H, d); 8.67 (2H, d); 9.07 (2H, d).

IR (KBr); 1741, 1725, 1255, 1144 cm⁻¹ (C═O and C--O).

EXAMPLE 266,6"-Bis(methoxycarbonyl)-4,4"-bis(4-nitrophenyl)-2,2';6',2"-terpyridine,17b

Compound 17b was synthesized from 16b using a method analogous to thesynthesis described in 17a.

Yield: 45%

¹ H NMR (CDCl₃): 4.05 (6H, s); 7.80-9.03 (15H, m).

IR (KBr): 1740, 1250, 1125 cm⁻¹ (C═O and C--O), 1525, 1345 cm⁻¹ (NO₂).

EXAMPLE 276,6"-Bis(methoxycarbonyl)-4,4"-bis(3-nitrophenyl)-2,2';6',2"-terpyridine,17c

Compound 17c was synthesized from 16c using a method analogous to thesynthesis described in 17a.

Yield: 38%

¹ H NMR (CDCl₃): 4.05 (3H, s); 4.09 (3H, s); 4.09 (3H, s); 7.75-8.97(15H, m)

IR (KBr): 1725, 1260, 1130 cm⁻¹ (C═O and C--O), 1530, 1340 cm⁻¹ (NO₂).

EXAMPLE 28 6,6"-Bis(hydroxymethyl)-4,4"-diphenyl-2,2';6',2"-terpyridine,18a

Sodium borohydride (0.13 g, 3.5 mmol) was added to a suspension of 17a(0.40 g, 0.8 mmol) in absolute ethanol (10 ml). After stirring for 2hours at room temperature the mixture was heated to reflux for 15 hours.The solution was evaporated in vacuo. A saturated solution of sodiumhydrogen carbonate (5 ml) was added to the residue. After the solutionwas brought to the boil, water (15 ml) was added. The mixture wasallowed to stand overnight in the cold. The precipitate was filtered,dried in air and finally crystallized from methanol.

Yield: 47%

m.p. 218°-219° C.

¹ H NMR (DMSO): 4.75 (4H, s); 7.52-7.95 (10H, m); 7.86 (2H, d); 8.14(1H, t); 8.49 (2H, d); 8.81 (2H, d).

EXAMPLE 296,6"-Bis(hydroxymethyl)-4,4"-bis(4-nitrophenyl)-2,2';6',2"-terpyridine,18b

Compound 18b was synthesized from 17b using a method analogous to thesynthesis described in 18a.

Yield: 40%

EXAMPLE 306,6"-Bis(hydroxymethyl)-4,4"-bis(3-nitrophenyl)-2,2';6',2"-terpyridine,18c

Compound 18c was synthesized from 17c using a method analogous to thesynthesis described in 18a.

Yield: 56%

¹ H NMR (DMSO): 4.98 (4H, s); 7.86-9.13 (15H, m)

IR (KBr): 1530, 1350 cm⁻¹ (NO₂).

EXAMPLE 31 6,6"-Bis(bromomethyl)-4,4"-diphenyl-2,2';6',2"-terpyridine,19a

A solution of phosphorous tribromide (0.12 g, 0.4 mmol) in chloroform (1ml) was added to a solution of 18a (0.13 g, 0.3 mmol) in chloroform (17ml). The mixture was refluxed for 11 hours whereafter the mixture wasneutralized with 5% sodium hydrogen carbonate. The aqueous layer wasextracted with chloroform (3×20 ml). The combined organic phase wasdried with sodium sulfate and evaporated in vacuo. The residue wascrystallized from dichloromethane.

Yield: 29%

m.p. 253.5°-255.5° C.

¹ H NMR (CDCl₃): 4.73 (4H, s); 7.47-7.81 (10H, m); 7.73 (2H, d); 8.01(1H, t); 8.57 (2H, d); 8.79 (2H, d).

EXAMPLE 32 6,6"-Bis(bromomethyl)-4,4"-bis(4-nitrophenyl)-2,2';6',2"-terpyridine, 19b

Compound 19b was synthesized from 18b using a method analogous to thesynthesis described in 19a.

Yield: 37%

₁ H NMR (CDCl₃): 4.75 (4H, s); 7.40-8.70 (15H, m).

EXAMPLE 336,6"-Bis(bromomethyl)-4,4"-bis(3-nitrophenyl)-2,2';6',2"-terpyridine,19c

Compound 19c was synthesized from 18c using a method analogous to thesynthesis described in 19a.

Yield: 50%

¹ H NMR (CDCl₃): 4.78 (2H, s); 4.83 (2H, s); 7.48-8.65 (15H, m)

EXAMPLE 346,6"-Bis[N,N-bis(carboxymethyl)aminomethyl]-4,4"-diphenyl-2,2';6',2"-terpyridine,20a

Potassium carbonate (120 mg, 0.9 mmol) was added to a mixture of 19a (47mg, 0.08 mmol) and di-t-butyl iminodiacetate (41 mg, 0.20 mmol) in dryacetonitrile (5 ml), and the mixture was stirred for 24 hours at roomtemperature. The mixture was evaporated in vacuo, the residue wasstirred with chloroform (10 ml), washed with water (2×10 ml) and driedwith sodium sulfate. Evaporation gave an oil which was characterized byNMR spectrum. The oil was dissolved in trifluoroacetic acid (3 ml) andkept at room temperature for 16 hours. The trifluoroacetic acid wasevaporated in vacuo, the solid residue was triturated with ethyl ether.

Yield: 59%

¹ H NMR (DMSO): 3.71 (8H, s); 4.27 (4H, s); 7.54-7.96 (10H, m); 8-04(2H, d); 8.18 (1H, t); 8.51 (2H, d); 8.85 (2H, d)

IR (KBr): 1734, 1630, 1395, 1200 cm⁻¹ (C═O and C--O).

EXAMPLE 356,6"-Bis[N,N-bis(carboxymethyl)-aminomethyl]-4,4"-bis(4-aminophenyl)-2,2';6',2"-terpyridine,20b

The reaction between 19b (0.11 g, 0.2 mmol) and diethyl iminodiacetate(76 mg, 0.40 mmol) in dry acetonitrile (10 ml) and potassium carbonate(0.27 g, 20 mmol) was made in analogy with 20a. The reaction gave an oilwhich was purified with flash chromatography (silica, MeOH/CHCl₃ 1/19).The oil was dissolved in methanol (5 ml). 10% Palladium on carbon (20mg) was added followed by slow addition of sodium borohydride (23 mg,0.60 mmol). After one hour the mixture was filtered and the filtrate wasevaporated in vacuo. The residue was dissolved in 0.5M potassiumhydroxide ethanol (20 ml). After stirring for 3 hours at roomtemperature the mixture was evaporated and water (20 ml) was added. Themixture was acidified with 6M HCl (pH about 2.0). The product wasfiltered and washed with water.

Yield: 60%

¹ H NMR (DMSO): 3.71 (8H, s); 4.30 (4H, s); 7.75-8.80 (15H, m)

IR (KBr): 1735, 1630, 1400, 1200 cm⁻¹ (C═O and C--O).

EXAMPLE 366,6"-Bis[N,N-bis(carboxymethyl)-aminomethyl]-4,4"-bis(3-aminophenyl)-2,2';6',2"-terpyridine,20c

Compound 20c was synthesized from 19c using a method analogous to thesynthesis described in 20b.

Yield: 79%

¹ H NMR (DMSO): 3.68 (8H, s); 4.21 (4H, s); 7.63-8.64 (15H, m)

IR (KBr): 1725, 1630, 1400, 1190 cm⁻¹ (C═O and C--O).

EXAMPLE 37 L-Lysine ethyl ester (starting compound, Scheme 3)

Thionyl chloride (5.0 ml, 8.06 g, 68 mmol) was added dropwise to 500 mlof ice- cooled dry ethanol. The stirred mixture was kept for 20 min atthis temperature and L-lysine hydrochloride (20 g, 109 mmol) was added.

The mixture was then refluxed for 3 h and concentrated to a volume ofabout 200 ml. 200 ml of diethylether was added and the crystallizedproduct filtered off.

Yield: 29 g (97%)-dihydrochloride. Rf=0.20 (System F)

EXAMPLE 38 ω-N-(4-Nitrobenzoyl)-L-lysine ethyl ester (37)

L-lysine HCl (5 g, 27.4 mmol) dissolved in 50 ml of water was titratedwith 5M NaOH to pH 10.5. 4-Nitrobenzoyl chloride (6.6 g, 36 mmol) indioxane (50 ml) and 5M NaOH were slowly added keeping the vigorouslystirred reaction mixture at pH 10.5.

After complete addition and disappearance of the pink colour thereaction mixture was acidified with conc. HCl to pH 2 and extracted fourtimes with diethylether. The aqueous phase was concentrated to dryness,coevaporated twice with 200 ml of dry ethanol and suspended in 250 ml ofdry ethanol previously treated with 10 ml of thionyl chloride. Themixture was refluxed for 3 h, filtered and evaporated. The residualmaterial was partitioned between saturated sodium bicarbonate andchloroform/ethanol 1:1 and the organic phase was dried over magnesiumsulfate yielding a crude product which was purified by flashchromatography using 5% EtOH/chloroform as eluent.

Yield: 1.08 g (12%) oil crystallizing on standing Rf=0.23 (System A)

H¹ NMR (60 MHz, CDCl₃): 8.25 (d,2H, J=9 Hz), 7.93 (d, 2H, J=9 Hz), 6.87(s, broad, 1H), 3.99-4.34 (q, 2H), 3.30-3.60 (m, 3H), 1.40-1.75 (m, 8H),1.11-1.37 (t, 3H)

EXAMPLE 39 α-N-(Methoxycarbonylmethyl)-ω-N-(4-nitrobenzoyl)-L-lysineethyl ester (38)

Compound (37) (0.54 g, 1.7 mmol) was coevaporated with toluene,dissolved in dry acetonitrile (10 ml) and bromoacetic acid methylester(0.265 g, 1.7 mmol) was added followed by pulverized dry sodiumcarbonate (2.0 g). The mixture was refluxed for 3 h.

Filtration of the inorganic salts and evaporation of the acetonitrilegave an oily crude product which was purified by flash chromatography.

Yield=0.45 g (68%) oil Rf=0.26 (System A)

H¹ NMR (60 MHz, CDCl₃): 8.25 (d, 2H, J=9 Hz), 7.93 (d, 2H, J=9 Hz), 6.63(s, broad, 1H), 3.95-4.30 (q, 2H), 3.68 (s, 3H), 3.30-3.60 (m, SH),1.40-1.75 (m, 7H), 1.11-1.37 (t, 3H).

EXAMPLE 40 ω-N-Monomethoxytrityl-L-lysine ethyl ester (39)

Dry triethylamine (1.8 ml, 18 mmol) was added to a suspension of thestarting compound (scheme 3) (1.5 g, 6 mmol) in 20 ml of dry pyridine.To this mixture stirred at RT, solid monomethoxytrityl chloride (1.96 g,6 mmol) (MMTrCl) was added in small portions during a period of 1 hwhereupon the mixture was stirred for additional 2 h. A standard sodiumbicarbonate work-up, followed by extraction with chloroform, yielded acrude product contaminated with α-MMTr isomer.

The pure title product was easily isolated by flash columnchromatography due to the large Rf difference between the isomers.

Yield: 1.35 g (48%) oil Rf=0.43 (System A)

H¹ NMR (400 MHz, CDCl₃): 7.5-6.75 (m, 14H), 4.18-4.13 (q, 2H), 3.78 (s,3H), 3.45-3.37 (m, 1H), 2.14-2.10 (t, 2H, J=7 Hz), 1.75-1.35 (m, 9H),1.26 (t, 3H)

EXAMPLE 41 α-N-(Methoxycarbonylmethyl)-ω-N-monomethoxytrityl-L-lysineethyl ester (40)

A partially protected L-lysine derivative (39) (1.0 g, 2.13 mmol) wasconverted to product (40) using the method described in Example 36.

Yield: 0.81 g (70%) oil Rf=0.73 (System A)

H¹ NMR (400 MHz, CDCl₃): 7.46-6.77 (m, 14H), 4.19-4.14 (q, 2H), 3.77 (s,3H), 3.70 (s, 3H), 3.31-3.45 (q, 2H), 3.22-3.25 (t, 1H), 2.09-2.12 (t,2H), 1.35-1.70 (m, 6H), 1.23-1.27 (t, 3H)

EXAMPLE 42 ω-N-Trifluoroacetyl-L-lysine ethyl ester (41)

The starting compound (scheme 3) (2.0 g, 8.1 mmol) dissolved in 10 ml ofdry ethanol was treated with dry triethylamine (4.09 g, 40.4 mmol).Ethyl trifluoroacetate (1.5 g, 10.5 mmol) was added to the stirredsuspension formed, and the mixture was refluxed for 6 h.

All volatiles were then evaporated and the residue was partitionedbetween saturated sodium hydrogen carbonate and chloroform/ethanol 1:1.

The combined organic phase (5×60 ml) was evaporated, coevaporated withtoluene and flash chromatographed to give the title product in the formof a colorless oil.

Yield: 1.9 g (87%) Rf=0.72 (System D)

H¹ NMR (400 MHz, CDCl₃): 7.10 (t, 1H, exchangeable), 4.21-4.16 (q, 2H),3.45-3.40 (m, 1H), 3.38-3.31 (m, 2H), 1.84 (s, 2H, exchangeable),1.82-1.40 (m, 6H), 1.28 (t, 3H).

EXAMPLE 43 α-N-(Methoxycarbonylmethyl)-ω-N-trifluoroacetyl-L-lysineethyl ester (42)

L-lysine derivative (41) (1.0 g, 3.7 mmol) was converted to the product(42) by means of a method analogous to Example 36.

Yield: 1.05 g (83%) oil Rf=0.48 (System A)

H¹ NMR (60 HMz, CDCl₃ +CD₃ OD): 4.4-4.0 (q, 2H), 3.68 (s, 3H), 3.5-3.1(m, 5H), 1.8-1.4 (m, 6H), 1.23 (t, 3H).

EXAMPLE 44 ω-N-(4-Hydroxybutyryl)-L-lysine ethyl ester (43)

L-lysine etyl ester×2 NCl (36) (2 g, 8.1 mmol) in 30 ml of dry ethanolwas treated with dry triethylamine (5.63 ml, 40.5 mmol) andγ-butyrolactone (0.7 g, 8.1 mmol) and the resultant suspension wasrefluxed for 3 h.

Evaporation of volatiles and coevaporation with toluene yielded a crudeproduct which was purified by flash chromatography using 20%methanol/chloroform as solvent.

Yield: 1.54 g (73%) oil Rf=0.28 (System D)

1 H¹ NMR (400 MHz, CDCl₃ +CD₃ OD): 4.30-4.22 (q, 2H), 3.72-3.77 (m, 1H),3.58-3.65 (t, 2H), 3.18-3.28 (m, 2H), 2.30-2.36 (t, 2H), 1.40-2.00 (m,8H), 1.28-1.34 (t, 3H).

EXAMPLE 46 Fluorescence of europium and terbium chelates of compound 20a

The relative fluorescence yield φ_(rel) of the europium and terbiumchelates of compound 20a were measured in equimolar 10⁻⁵ M solutions ofcompound 20a and the corresponding lanthanide ion. Fluorescencemeasurements were done on a Perkin-Elmer LS-5® spectrofluorometer usingthe phosphorescence mode which allowed the decay curves of thelanthanide fluorescence to be measured. The fluorescence yield isreported relative to the fluorescence of the uncomplexed lanthanidecation (Ln) using the equation: ##EQU1## where I_(che) and I_(Ln) arethe preexponential terms of the emission decay curves for the chelatedand uncomplexed lanthanide cation, respectively (614 nm for europium and544 nm for terbium). The excitation wavelength for the uncomplexedeuropium was 395 nm and for terbium 370 nm. C_(Ln) and C_(che) are theconcentrations of free and complexed lanthanide cation, respectively,and k_(Ln) and k_(che) the corresponding decay constants. For compound20a the relative fluorescence yield for the europium comples becomes2.1×10⁶ and for the terbium complex 8.1×10⁴. The excitation wavelengthfor the chelates was in both cases 340 nm.

We claim:
 1. A lanthanide chelate in which a lanthanide selected fromthe group consisting of Eu³⁺, Tb³⁺, Sm³⁺ and Dy³⁺ is chelated to aterpyridine compound having the structure ##STR4## wherein (i) n=1 or2,(ii) X₁ =X₅ hydrogen, (iii) X₂, X₃ and X₄ are selected from the groupconsisting of hydrogen, hydroxy, amino, amido, alkoxy, alkyl, alkynyl,aryl, aralkyl, aryloxy, arylamino and arylethynyl, with the proviso thatalkyl has less than 12 carbon atoms and aryl is selected from phenyl,quinolyl, naphthyl, pyridyl and pyrimidyl, (iv) Ch is a chelating groupselected from --COO⁻, --OPO₃ ²⁻ and- PO₃ ²⁻, (v) X-Y is a substituentreplacing a group X₁ -X₅ or H', in which X is a bridge hot participatingin the chelating and consisting of a pure hydrocarbon chain of 1-12carbon atoms and at least one structural element selected from the groupconsisting of secondary and tertiary amines, aliphatic disulfides,aliphatic thioethers, ethers, esters and diaza, and Y is a residue of acompound selected from the group consisting of antigens, haptens,antibodies and nucleic acids, said residue retaining the biospecificaffinity of said compound.
 2. A chelate according to claim 1 wherein thepure hydrocarbon chain is selected from the group consisting of phenyland naphthyl.
 3. A chelate according to claim 1 wherein n=1; X₂ and X₄are hydrogens; Ch is --COO⁻ ; X-Y is 4-aminophenyl to which the residueY is linked at the 4-amino group; and X-Y replaces X₃.
 4. A chelateaccording to claim 1 wherein n=1; X₂ and X₄ are hydrogens; Ch is --COO⁻; X-Y is 3-aminophenyl to which the residue Y is linked at the 3-aminogroup; and X-Y replaces X₃.
 5. A chelate according to claim 1 whereinn=2; X₃ is hydrogen; Ch is --COO⁻ ; X-Y is 4-aminophenyl to which theresidue Y is linked at the 4-amino group; and X-Y replaces X₂ and X₄,respectively.
 6. A chelate according to claim 2 wherein n=2; X₃ ishydrogen; Ch is --COO⁻ ; X-Y is 3-aminophenyl to which the residue Y islinked at the 3-amino group; and X-Y replaces X₂ and X₄, respectively.7. A chelate according to claim 2 wherein n=1; X₂ and X₄ are hydrogens;X-Y is isothiocyanatophenyl to which the residue Y is linked at theisothiocyanato group; and X-Y replaces X₃.